In 1982, a Canadian patent No. 1,120,399 entitled "Pharmaceutical composition for treatment of tumor cells" by Nigam, Vijai N. and Brailovsky, Carlos, A. was granted to Universit e de Sherbrooke. It described the administration of certain fatty acid esters of mono and disaccharides which surprisingly elicit an antitumor response as shown by an enhancement of the host capacity to reject a large number of tumor cells, to retard growth in tumor size and to induce hemorrhagic tumor necrosis. Fatty acid esters of maltose, galactose, glucose, mannose, arabinose, cellobiose and lactose were particularly useful when the fatty acid comprised 12 to 18 carbon atoms.
At that time the method employed for the preparation of these compounds was not patented since it consisted in a conventional methodology without regards to providing stringent purity requirement and optimal yields. The method that was based on the deployment of a known solvent system used for thin layer chromatography (TLC) on silica gel G plates. It employed a silica gel column instead of silica gel plates, thereby avoiding scraping of bands from plates, and obtained larger amounts of the product. A fraction isolated from the column that gave a thick band of Rf 0.68 on TLC plates and a ratio of glucose to palmitic acid of approximately 0.5 was referred to as maltose tetrapalmitate. The compound prepared accordingly was used for numerous biological experiments.
It was further surprisingly noted that the fraction referred to as maltose tetrapalmitate and used in biological investigation was indeed not pure and, upon rechromatography on TLC plates in other solvent systems, provided 2-3 bands.
In addition, it was further noted that the band of Rf 0.68 in CHCl.sub.3 : MeOH:H.sub.2 O (60:25:4) solvent system, upon scraping from unstained silica gel plates could be split into 3 bands upon rechromatography in other solvent systems as well. In addition, it contained small amounts of palmitic acid. These results which came as a surprise established that there may be an association between the three components due to hydrophobic interaction because of the presence of water in the solvent system which was comprised of CHCl.sub.3 :MeOH:H.sub.2 O, 60:25:4. To our satisfaction, the property of hydrophobic interactions between various bacterial lipid As (which are structurally similar to maltose palmitates) and the splitting of single bands upon rechromatography had been described before (Chen et al. J. Infect. Dis. 128: 543-551, 1973).
The question confronting us was to find the identity of the three components and to find out the most active antitumor component among them, characterize it, and to see if associating them in various ratios provided a more active product than individual components as far as its antitumoral activity in vivo and its solubility in aqueous solvent were concerned. To date, we have seen no report for the isolation of pure fractions of maltose palmitates which have been structurally well characterized and have been tested for their antitumor activity. One report based on our initial finding described antitumor activity of maltose mono fatty acyl esters which was superior to maltose poly fatty acyl esters but the components were not well characterized and the purification procedure used was column chromatography similar to the one used by us (Nishikawa, Y., Yoshimoto, K., Nishijima, M., Fukuoka, F. and Ikekawa, T. Chem. Pharm. Bull. 29 (2): 505-513, 1981). Mono esters are mild detergents and could lyse tumor cells at the high concentrations used by those researchers.
Another remarkable finding made by us (Anticancer Res. 9:1883-1888, 1989) was that a combination of crude MTP with cortisone or .alpha.-OH progesterone or tetrahydro S resulted in a high antitumor activity and it was interpreted as being caused by the antiangiogenic activity of the combination, rather than immune stimulating activity, since hydrocortisone is known to be highly immunosuppressive.
It should be noted that both the Health Protection Branch (HPB) in Canada and FDA in U.S.A. insist on the use of characterized products for human use, especially when they are prepared by synthetic routes. The use of uncharacterized mixtures alone or in formulations is not permitted. The major problem in the use of uncharacterized partially purified mixtures of substances is badge variation and the presence of impurities that may remain associated within the mixture and elicit toxic reactions on dose escalation and upon chronic use. In our case, once it became apparent that our column prepared MTP (as described in our previous patent) was not a single component, there was no excuse for not identifying and characterizing the individual components and finding which was the active one. Indeed, as detailed later, it became clear that the most active component was maltose tripalmitate, rather than maltose tetrapalmitate (as previously thought) and that maltose tetrapalmitate could not even be administered due to its lack of solubility. Maltose tetrapalmitate when emulsified could be injected intraperitoneally (ip) and its activity was derived from its transformation to the maltose tripalmitate. Thus the claim in our earlier patent stating that the active component was maltose tetrapalmitate was only partly true.
It seemed to us that:
1) new methods of purification must be devised using HPLC, which would have a high capacity of resolution and which can be adapted for future large scale isolation of the components, especially with the new pilot plant HPLC separation equipment provided by Waters Inc.;
2) the activities of purified components, individual maltose palmitates, should be tested in the presence and absence of hydrocortisone to see if their activity is based on immunological stimulation, or based on antiangiogenic activity;
3) the active antitumor agents should be structurally characterized after their separation on HPLC using chemical means; and
4) a pharmaceutical formulation should be made to take into account the distribution of individual maltose palmitates in various organs, the rate of degradation of higher palmitate esters into lower palmitate esters, and a good solubility or dispersibility. This would lead to suggested optimum dose and/or delivery rate devised specifically for cancers of different organs.
The isolation of the three individual products contained in the previously described MTP was attained in a long and painstaking manner. The use of HPLC as a chromatographic tool for separation of closely related substances is well known and the resolving power of HPLC surpasses those of other chromatographic techniques. However, finding the appropriate support systems and solvents requires numerous trials to arrive at the most suitable combinations. Up to date, to our knowledge, fatty acyl esters of sugar have not been subjected to rigorous separation and few, if any, have been structurally characterized with respect to the position of esterification. Most characterizations end up solely with the number of fatty acid residue per mono or disaccharide molecule.